Our immunoblot analysis suggested that REEP1 protein expression was restricted, since several different cell lines demonstrated no expression of REEP1 (HEK293/HEK293A, Rat1, PC12, NRK). both immunoblotting and immunofluorescent microscopic analysis. Unlike previous RT-PCR studies, immunoblotting demonstrated that REEP1 protein was not ubiquitous; its expression was restricted to neuronal tissues (brain, spinal cord) and testes. Gene expression microarray analysis demonstrated REEP1 and REEP2 mRNA expression in superior cervical and stellate sympathetic ganglia tissue. Furthermore, expression of endogenous REEP1 was confirmed in cultured murine sympathetic ganglion neurons by RTPCR and immunofluorescent staining, with expression occurring between Day 4 and Day 8 of culture. Lastly, we demonstrated that REEP2 protein expression was also restricted to neuronal tissues (brain and spinal cord) and tissues that exhibit neuronal-like exocytosis (testes, pituitary, and adrenal gland). In addition to sensory tissues, expression of the REEP1/REEP2 subfamily appears to be restricted to neuronal and neuronal-like exocytotic tissues, consistent with neuronally restricted symptoms of REEP1 genetic disorders. Keywords:receptor expression enhancing protein, neurodegeneration, sympathetic ganglion neuron, hereditary spastic M?89 paraplegia, distal M?89 hereditary motor neuropathy type V == 1. Introduction == Many neuronally relevant G protein-coupled receptors (GPCRs) have proven difficult to express in heterologous cell systems (e.g. HEK293), including olfactory (ORs), taste (TRs), and vomeronasal (VRs) receptors (Behrens et al., 2006;Dulac and Axel, 1995;Mombaerts, 2004). Similar to ORs and TRs, 2C adrenergic receptors (ARs) are also not easily expressed in nonneuronal cells, however, 2A ARs do not show such difficulty. However, heterologous expression of 2C ARs in neuronal cell lines (e.g. PC12 cells) led to significant plasma membrane expression, suggesting a role for a neuronal-specific accessory protein(s) (Angelotti et al., 2010;Hurt et al., 2000). M?89 Furthermore, 2A and 2C ARs show differential localization within cultured sympathetic ganglion neurons (SGN) (Brum et al., 2006). It was hypothesized that neuronal or sensory cells may express cell-specific accessory M?89 proteins necessary for proper membrane targeting of these GPCRs. While attempting to identify possible accessory proteins, Matsunami and colleagues identified two new families of proteins that appeared to enhance surface expression of ORs, the Receptor Expression Enhancing Protein (REEP) and Receptor Transporting Protein (RTP) families (Saito et al., 2004). In total, six REEPs and four RTPs were identified. The REEP family can be subdivided into two subfamilies REEP14 and REEP56, with the latter showing the most homology with the yeast homolog Yop1 (Park et al., 2010). Evolutionary analysis has suggested Rabbit Polyclonal to NMS that REEP14 evolved from two rounds of whole genome duplication, starting with creation of REEP1/2 and REEP3/4 subfamilies, and eventually REEPs 14 (Tinti et al., 2012). Thus, it is plausible that REEP1 and REEP2 evolved to serve tissue-specific functions. RTP homologs have not been identified outside of vertebrates; however, REEPs belong to a much larger Yip (Ypt interacting protein) family, which has been conserved in invertebrates, yeast, plants, and mammals (Pfeffer and Aivazian, 2004;Tinti et al., 2012). Various Yip family members have been shown to interact directly with M?89 Rab GTPases and ER/Golgi vesicle proteins to regulate intracellular trafficking and targeting of cargo proteins within yeast and neurons (Al Awabdh et al., 2012;Brands and Ho, 2002;Calero et al., 2001;Carrel et al., 2008;Heidtman et al., 2003;Martincic et al., 1997). Recent work has demonstrated that REEP isoforms are important determinants of ER tubular structure (Park et al., 2010;Voeltz et al., 2006) and can be further described as ER membrane shaping adapter proteins, based upon their interactions with 14-3-3 proteins and GPCRs, such as 2C ARs (Bjrk et al., 2013;Johnson et al., 2011). The recent discovery that multiple REEP1 mutations are linked to the neurodegenerative disorders hereditary spastic paraplegia (HSP) and distal hereditary motor neuropathy type V (dHMN-V) has further increased interest in this family of proteins (Beetz et al., 2008;Beetz et al., 2012;Zuchner et al., 2006). Northern blot,in situhybridization, RT-PCR, and immunofluorescent analysis has determined REEP expression patterns in various tissues, often with conflicting results. Consistent with enhancement of OR and TR expression, various isoforms were found to be expressed in olfactory and vomeronasal epithelium, circumvallate papillae (tongue), brain, and cultured cortical neurons (Behrens et al., 2006;Ilegems et.